E1-E2-E3 enzyme-mediated covalent attachment of ubiquitin (Ub) or ubiquitin-like proteins (Ubls) such NEDD8 is a predominant form of eukaryotic protein regulation. Ubls modify a vast number of proteins and alter their functions in a variety of ways. For example, Ub/Ubl modifications can affect the target protein's half-life, subcellular localization, enzymatic activity, or ability to interact with protein or DNA partners. As a result, Ub/Ubls regulate numerous biological processes, such as the cell cycle, signal transduction, apoptosis, the immune response, autophagy, and development. Defects in Ub/Ubl pathways are widely associated with diseases, including cancers, developmental disorders, high blood pressure, neurodegenerative disorders, and cachexia. We propose to extend our expertise on Ub/Ubl pathways to mechanisms of ligation by the three largest E3 families: HECT (Homologous to E6AP C-Terminus - 28 predicted in humans), RING (Really Interesting New Gene - 570 predicted in humans) and RBR (RING1-IBR-RING2 - 13 predicted in humans). HECT and RBR E3s participate directly in catalysis, with a catalytic cysteine that forms a covalent intermediate through thioester-linkage with Ub's C-terminus via a 2-step reaction. First, the HECT or RBR E3 binds a thioester-linked E2~Ub intermediate, and Ub is transferred from the E2 catalytic cysteine to an E3 catalytic cysteine. Second, Ub is transferred from the E3 Cys to a target lysine. By contrast, RING E3s promote transfer of Ub or a Ubl from from an E2~Ub/Ubl intermediate to an associated substrate. Among the RING E3s, the largest class consists of the modular, multisubunit Cullin-RING (CRL) family. CRLs function sequentially with distinct E2s to modify distinct targets: first the RING domain binds an E2~NEDD8 intermediate, and the cullin subunit is activated by self-modification with NEDD8. Then a NEDD8-modifed CRL binds a Ub-loaded E2, which is the source of Ub to be transferred to a target. Ultimately, for all three classes of E3, repeated cycles through their enzymatic cascades can lead to polyubiquitination, with specific enzymes selectively linking a donor Ub's C-terminus to distinctive lysines on the acceptor Ub's surface. We propose a research plan focused on structural biology and biochemistry to understand mechanisms underlying Ub/Ubl ligation, determining target specificity, and modulating functions of HECT (Aim 1), CRL (Aim 2), and RBR E3s (Aim 3).